PROJECT SUMMARY Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD) are severe neurodegenerative diseases that affect more than 30,000 people in the United States. ALS/FTLD, which exist on a pathological spectrum, is hallmarked by the progressive loss of neurons. This leads to defects in motor neuron function and eventually death, and there is no known cure for ALS/FTLD. A key molecular phenotype of ALS/FTLD is the aggregation of proteins such as Fused in sarcoma (FUS), an RNA-binding protein with over 70 mutations linked to ALS/FTLD. Importantly, FUS undergoes liquid-liquid phase-separation (LLPS) to form condensates that likely contribute to its functions in RNA metabolism and the DNA damage response. However, this normally-reversible LLPS is altered by Fus mutations, which instead favor a sol-gel transition that forms aggregates that are toxic to neurons. This aggregation drives ALS/FTLD in patients. FUS mutations are heterozygous, indicating that this pathological phenotype occurs despite the presence of wild-type FUS. How mutant FUS interacts with wild-type FUS ? if at all ? is unknown. Wild-type FUS may decelerate progression of FUS-driven ALS/FTLD by breaking apart mutant FUS aggregates. As mutant FUS ages, it might escape recovery by wild-type FUS (gain-of-toxicity). Alternatively, wild-type FUS might get trapped in pathogenic aggregates (loss-of-function). Investigating the interactions between wild-type and mutant FUS is critically important for understanding the molecular mechanism underlying ALS/FTLD, and for developing effective treatments. Therefore, I propose to interrogate the wild-type/mutant FUS interaction in vitro and in vivo with single-molecule techniques. I plan to address three fundamental questions: (1) Do wild-type and mutant FUS interact? (2) What is the RNA binding phenotype of mixed wild-type and mutant FUS? (3) How does mutant FUS aging alter wild-type/mutant interactions? I will use a combination of single-molecule assays (single-molecule FRET, single-molecule FUS nucleation, and single- molecule tracking) and ensemble assays (in vitro droplet formation, RNA splicing, and droplet fusion by optical tweezers). This proposal is strengthened by contributions from four collaborators (see support letters), all of whom are experts on ALS or LLPS. These collaborations and other professional development opportunities will drive my preparation for a postdoctoral position. I will also focus on improving my scientific communication, and I plan to become an expert on single-molecule techniques to uniquely position myself for my future academic career. Altogether, this proposal will develop and use single-molecule techniques to better understand the early steps of ALS pathogenesis.